Analysis of mutations in the pore-forming region essential for insecticidal activity of a Bacillus thuringiensis delta-endotoxin

The Bacillus thuringiensis insecticidal delta-endotoxins have a three-domai n structure, with the seven amphipathic helices which comprise domain I bei ng essential for toxicity. To better define the function of these helices i n membrane insertion and toxicity, either site-directed or random mutagenes is of two regions was performed. Thirty-nucleotide segments in the B. thuri ngiensis crylAc1 gene, encoding parts of helix alpha 4 and the loop connect ing helices alpha 4 and alpha 5, were randomly mutagenized, This hydrophobi c region of the toxin probably inserts into the membrane as a hairpin. Site -directed mutations were also created in specific surface residues of helix alpha 3 in order to increase its hydrophobicity. Among 12 random mutations in helix alpha 4, 5 resulted in the total loss of toxicity for Manduca sex ta and Heliothis virescens, another caused a significant increase in toxici ty, and one resulted in decreased toxicity, None of the nontoxic mutants wa s altered in toxin stability, binding of toxin to a membrane protein, or th e ability of the toxin to aggregate in the membrane. Mutations in the loop connecting helices alpha 4 and alpha 5 did not affect toxicity, nor did mut ations in alpha 3, which should have enhanced the hydrophobic properties of this helix. In contrast to mutations in helix alpha 5, those in helix alph a 4 which inactivated the toxin did not affect its capacity to oligomerize in the membrane, Despite the formation of oligomers, there was no ion flow as measured by light scattering. Helix alpha 5 is important for oligomeriza tion and perhaps has other functions, whereas helix alpha 4 must have a mor e direct role in establishing the properties of the channel.